INAUGURAL ARTICLE by a Recently Elected Academy Member:Integrins α1β1 and α2β1 are receptors for the rotavirus enterotoxin

Rotaviruses (RVs) are nonenveloped, triple-layered icosahedral viruses containing a genome of 11 segments of dsRNA that code for six structural (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural (NSP1–NSP6) proteins (1). RV is the leading etiologic agent of severe gastroenteritis in infants, young children, and animals worldwide. There is no known treatment to prevent RV transmission. Although new vaccines have recently shown promising results in clinical trials and are now licensed (2), vaccine efficacy in developing countries where disease prevention is needed most remains unknown. The recent recognition that RV causes extraintestinal infection in animals and children (3–6) highlights a continuing need to better understand the mechanisms of RV replication and pathogenesis. Increasing knowledge of the structure of RVs and protein function has supported new ideas about virus pathogenesis, virus assembly, and virus–host interactions (1, 7, 8). However, the molecular details of RV effects on intestinal epithelial cells or extraintestinal cells remain incompletely understood. NSP4 is the first described viral enterotoxin (9–11). The i.p. delivery of purified recombinant NSP4 or synthetic NSP4 peptides from mammalian and avian RVs that lack sequence similarity causes age-dependent diarrhea in young mice (9, 12–16). The cytoplasmic toxic domain of NSP4 (aa 112–175) is released from infected cells and hypothesized to interact with receptors on neighboring cells (9, 10, 17), mobilizing Ca2+ by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production (17), and eliciting age-dependent diarrhea in neonatal mice (9, 18). Integrins are a family of heterodimeric cell surface receptors composed of α and β subunits involved in cell–cell, cell–extracellular matrix, and cell–pathogen interactions (19–25). Vertebrates have nine α subunits that contain a so-called I or A domain, in which a set of α subunits (α1, α2, α10, and α11) act as collagen receptors (21). The I domain forms a subset of the larger group of von Willebrand factor A (VWA) domains found in a wide range of proteins (26), and many appear to be involved in protein–protein interactions. The x-ray structures of recombinant α1, α2, αM, and αL I domains have defined a divalent cation-binding motif [metal ion-dependent adhesion site (MIDAS)] that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands (27–30). The discovery that NSP4 functions as an enterotoxin and triggers epithelial cell responses stimulated a search for target molecule(s) that bind extracellular NSP4. Surface plasmon resonance (SPR) analysis isolated soluble HT29 surface components that bound to (His)6-NSP4FL immobilized onto a Ni2+-nitrilotriacetic acid (Ni2+-NTA)-coated sensor chip. Bound proteins were identified by mass spectrometry. Further biochemical, mutagenesis, and functional analyses identified two distinct functional domains on NSP4 (lacking known integrin-binding motifs) as being responsible for the binding to the MIDAS motif on α1 and α2 I and for eliciting C2C12-α2 spreading, respectively, and correlated with inducing diarrhea.